Perfusion of a human colon adenocarcinoma cell line, LS174T, on hollow fibers for one month produces tissue-like masses of cells with histopathology reminiscent of the patient's original tumor. Synthetic hollow fibers stimulate a vascular system through which low molecular weight nutrients and dissolved gases diffuse freely. Twelve clones of LS174T cells have been isolated and are being characterized rigorously. The criteria upon which the clones are evaluated include: 1) morphology, 2) carcinoembryonic antigen and mucin production, 3) tumor induction in nude mice, 4) colony formation in soft agar, 5) population doubling time, 6) isozyme profile, and 7) development of organoid growth in hollow fiber culture. Clones were established to identify the presence of pluripotential stem cells within the LS174T cell line that are capable of differentiation when cultured in the hollow fiber system. Recently one clone developed glands similar to the parental line in the hollow fiber system. The extra-capillary milieu is in contact with the growing cells, and the fluids contain moieties greater in molecular weight than the fiber pore size exclusion. We are studying the ability of these fluids to modulate cell:cell interactions during aggregation and three dimensional growth. Cells are exposed to extra-capillary fluids in either a rotating aggregation assay or in a soft agar matrix. The target cells include LS174T and clones or other human colon tumor cell lines with varying degrees of differentiation. Biological assays are used to monitor the presence of the factors during isolation and characterization. These studies may provide information on organotypic expression in vitro, which may be harnessed to effect phenotypic modulations of in vivo neoplasms, and may provide insights into cellular interactions and molecular triggers of differentiation of human epithelial tumor cells.